Identifying and utilizing the nutrients available in the most efficient manner is a challenge common to all organisms. In humans inaccurate or failed nutrient sensing can result in a variety of diseases including diabetes and obesity, and cancer progression has been shown to rely on increased glucose uptake and changes in nutrient sensing. Many of the nutrient sensing pathways are conserved from yeast to humans, and studies on nutrient sensing in unicellular eukaryotes have been instrumental in elucidating nutrient sensing pathways in humans. However, much of the work on nutrient signaling in unicellular eukaryotes has been done in budding yeast, which has a fairly limited carbohydrate utilization repertoire. Unlike budding yeast, the model filamentous fungus, Neurospora crassa, is capable of utilizing a wide variety of carbohydrates: from simple sugars to the complex sugar chains found in plant cell walls. In order to efficiently exploit the available resources, N. crass must be capable of sensing and responding to the presence of these different carbohydrates. Several transcription factors have been identified in N. crassa that activate the transcription of plant cell wall-degrading enzymes. One of these is XLR1, which activates the transcription of hemicellulases when in the presence of the plant cell wall component xylan. However, while it seems evident that XLR1 must be activated in order to induce expression of hemicellulases, the method by which this is achieved is still unclear. The goal of this project is to identify and characterize upstream regulators of XLR1 and their interactions with other nutrient sensing pathways in N. crassa, which will be accomplished through the completion of the following three specific aims. The first is to screen for mutants in which XLR1 activation is either constitutive o uninducible to identify the genes involved in the xylan-sensing pathway and characterize their function and role in xylan sensing. The second aim is to use directed evolution to probe more subtle genetic interactions both within the xylan-sensing pathway and between the xylan-sensing pathway and other nutrient sensing pathways to identify mutations which optimize N. crassa for rapid and accurate xylan sensing and utilization as well as to understand how the xylan-sensing pathway fits into the larger scheme of pathways that assess the metabolic state of the cell. And the third aim is to use synthetic biology to reconstruct the xylan-sensing pathway in the budding yeast, Saccharomyces cerevisiae, which is not able to use xylan as a carbon source, using the optimized genes identified in the first two aims to validate the identification o genes involved in the xylan-sensing pathway. The completion of these aims should shed light on the xylan-sensing pathway in N. crassa and its interaction with other cellular signaling pathways. We expect this to improve our overall understanding of cellular signaling throughout the eukaryotic realm , since scanning the diversity of molecular mechanisms involved in signaling pathways is helpful in elucidating broad biological paradigms.